Leonid V. Savitch

Photosystem II Excitation Pressure and Development of Resistance to Photoinhibition (II. Adjustment of Photosynthetic Capacity in Winter Wheat and Winter Rye)

Winter wheat (Triticum aestivum L. cv Monopol), spring wheat (Triticum aestivum L. cv Katepwa), and winter rye (Secale cereale L. cv Musketeer) grown at 5[deg]C and moderate irradiance (250 [mu]mol m-2 s-1) (5/250) exhibit an increased tolerance to photoinhibition at low temperature in comparison to plants grown at 20[deg]C and 250 [mu]mol m-2 s-1 (20/250). However, 5/250 plants exhibited a higher photosystem II (PSII) excitation pressure (0.32–0.63) than 20/250 plants (0.18–0.21), measured as 1 - qP, the coefficient of photochemical quenching. Plants grown at 20[deg]C and a high irradiance (800 [mu]mol m-2 s-1) (20/800) also exhibited a high PSII excitation pressure (0.32–0.48). Similarly, plants grown at 20/800 exhibited a comparable tolerance to photoinhibition relative to plants grown at 5/250. In contrast to a recent report for Chlorella vulgaris (D.P. Maxwell, S. Falk, N.P.A. Huner [1995] Plant Physiol 107: 687–694), this tolerance to photoinhibition occurs in winter rye with minimal adjustment to polypeptides of the PSII light-harvesting complex, chlorophyll a/b ratios, or xanthophyll cycle carotenoids. However, Monopol winter wheat exhibited a 2.5-fold stimulation of sucrosephosphate synthase activity upon growth at 5/250, in comparison to Katepwa spring wheat. We demonstrate that low-temperature-induced tolerance to photoinhibition is not a low-temperature-growth effect per se but, instead, reflects increased photosynthetic capacity in response to elevated PSII excitation pressure, which may be modulated by either temperature or irradiance.

Photometric method for routine determination of kcat and carbamylation of rubisco

Ribulose bisphosphate carboxylase (rubisco) is the first enzyme in photosynthetic CO2 assimilation. It is also the single largest sink for nitrogen in plants. Several parameters of rubisco activity are often measured including initial activity upon extraction, degree of carbamylation, catalytic constant of the enzyme (kcat), and the total amount of enzyme present in a leaf. We report here improvements of the photometric assay of rubisco in which rubisco activity is coupled to NADH oxidation which is continuously monitored in a photometer. The initial lag usually found in this assay was eliminated by assaying rubisco activity at pH 8.0 instead of 8.2, using a large amount of phosphoglycerate kinase, and adding monovalent cations to the assay buffer. We found that when using the photometric assay, the ratio of activity found initially upon extraction divided by the activity after incubating with CO2 and Mg2+ reflects the degree of carbamylation as determined by 14carboxyarabinitol bisphosphate/12carboxyarabinitol bisphosphate competition. We developed methods for measuring the catalytic constant of rubisco as well as the total amount of enzyme present using the photometric assay and carboxyarabinitol 1,5-bisphosphate. We believe that the photometric assay for activity will prove more useful than the 14CO2 assay in many studies.

Acclimation to low temperature or high light mitigates sensitivity to photoinhibition: roles of the Calvin cycle and the Mehler reaction

Winter wheat (Triticum aestivum L cv. Monopol) plants grown under either control (20˚C, 250 PFD), low temperature (5˚C, 250 PFD) or high light conditions (20˚C, 800 PFD) were compared in order to assess the roles of the Calvin cycle and the Mehler reaction in the differential sensitivity to chronic photoinhibition. Despite similar photosynthetic responses to irradiance, the partial pressure of CO2 [p(CO2)] and photoinhibition, photosynthetic acclimation to cold temperature appears to be quite distinct from acclimation to high light. First, the lower ratio of Rubisco oxygenation/Rubisco carboxylation and the reduced effects of p(CO2) on number of electrons per mole of CO2 fixed in cold-acclimated compared to high light-grown wheat indicate that photorespiration is differentially suppressed in cold-acclimated Monopol. Second, inhibition of the Calvin cycle by glyceraldehyde during photo-inhibition indicated that the sensitivity of high light-acclimated Monopol to photoinhibition was more dependent on Rubisco activity than the sensitivity of cold-acclimated plants to photoinhibition. Third, cold-acclimated Monopol exhibited higher electron transport rates in the presence of either ambient CO2, 2 kPa O2 or N2, 2 kPa O2 (either 77% or 68%, respectively) relative to controls compared to high light-acclimated plants exposed to similar gaseous environments (either 57% or 38%, respectively). Last, the activation state of NADP–malate dehydrogenase indicated that the stroma is highly reduced during cold acclimation relative to either controls or high light-grown Monopol. Thus, in cold-acclimated wheat, the Mehler reaction appears to play an important role while photorespiration plays a minimal role in mitigating the sensitivity to photoinhibition. In contrast, both photorespiration and the Mehler reac-tion appear to mitigate the sensitivity to photoinhibition in high light-grown Monopol. This is consistent with the differential sensitivity to methylviologen and the differential SOD activity observed between cold-acclimated and high light-grown Monopol.

Photosystem II Excitation Pressure and Photosynthetic Carbon Metabolism in Chlorella vulgaris

Chlorella vulgaris grown at 5[deg]C/150 [mu]mol m-2 s-1 mimics cells grown under high irradiance (27[deg]C/2200 [mu]mol m-2 s-1). This has been rationalized through the suggestion that both populations of cells were exposed to comparable photosystem II (PSII) excitation pressures measured as the chlorophyll a fluorescence quenching parameter, 1 - qP (D.P. Maxwell, S. Falk, N.P.A. Huner [1995] Plant Physiol 107: 687–694). To assess the possible role(s) of feed-back mechanisms on PSII excitation pressure, stromal and cytosolic carbon metabolism were examined. Sucrose phosphate synthase and fructose-1,6-bisphosphatase activities as well as the ratios of fructose-1,6-bisphosphate/fructose-6-phosphate and sucrose/starch indicated that cells grown at 27[deg]C/2200 [mu]mol m-2 s-1 appeared to exhibit a restriction in starch metabolism. In contrast, cells grown at 5[deg]C/150 [mu]mol m-2 s-1 appeared to exhibit a restriction in the sucrose metabolism based on decreased cytosolic fructose-1,6- bisphosphatase and sucrose phosphate synthase activities as well as a low sucrose/starch ratio. These metabolic restrictions may feed-back on photosynthetic electron transport and, thus, contribute to the observed PSII excitation pressure. We conclude that, although PSII excitation pressure may reflect redox regulation of photosynthetic acclimation to light and temperature in C. vulgaris, it cannot be considered the primary redox signal. Alternative metabolic sensing/signaling mechanisms are discussed.

Effects of growth under UVA radiation on CO2 assimilation, carbon partitioning, PSII photochemistry and resistance to UVB radiation in Brassica napus cv. Topas

The effects of growth of Brassica napus L. cv. Topas under PAR or PAR+UVA radiation was assessed with respect to sensitivity to subsequent exposure to UVB radiation. Despite the fact that growth under PAR+UVA induced minimal effects in photosystem II (PSII) photochemistry, growth under PAR+UVA inhibited the accumulation of the photosynthetic end products, sucrose and starch. This was associated with a decreased capacity for ribulose 1,5-bisphosphate (RuBP) regeneration, a decreased capacity for light- and CO 2 -saturated rates of CO2 assimilation, a decrease in the apparent quantum yield for CO2 assimilation, an over-reduction of chloroplast stroma, an increased susceptibility to the feedback effects on photosynthesis and a stimulation of glycolysis compared to controls grown under PAR. Subsequent exposure to UVB decreased the maximum Rubisco activity in leaves of both PAR- and PAR+UVA-grown plants. However, the decrease in the capacity for CO2 assimilation in PAR-grown plants exposed to UVB did not appear to be associated with limitations at the level of PSII linear electron transport, but rather with a decreased capacity for sucrose biosynthesis, limited triose-P utilization and a decreased capacity for RuBP regeneration. In contrast, the decreased capacity for CO 2 assimilation in PAR+UVA-grown plants exposed to UVB was associated with an inhibition of PSII photochemistry and a decreased supply of ATP. Thus, growth under UVA radiation appears to induce feedback-limited photosynthesis and does not enhance resistance of Brassica napus to UVB radiation.

Enhancing biomass production and yield by maintaining enhanced capacity for CO2 uptake in response to elevated CO2

Dahal, K., Weraduwage, S. M., Kane, K., Rauf, S. A., Leonardos, E. D., Gadapati, W., Savitch, L., Singh, J., Marillia, E.-F., Taylor, D. C., Micallef, M. C., Knowles, V., Plaxton, W., Barron, J., Sarhan, F., Hüner, N., Grodzinski, B. and Micallef, B. J. 2014. Enhancing biomass production and yield by maintaining enhanced capacity for CO2 uptake in response to elevated CO2. Can. J. Plant Sci. 94: 1075-1083. Using four model plants, two members of the Gramineae, rye and wheat, and two Brassicaceae, Brassica napus and Arabidopsis thaliana, two fundamental approaches were exploited to determine how regulating source-sink development would alter photosynthesis, productivity and yield during long-term acclimation to elevated CO2. In one approach we exploited the cold acclimation response of winter wheat, rye and B. napus. In the other approach we modified the dark respiration in A. thaliana to alter availability of respiratory substrates required for anabolic processes, such as fatty acid metabolism, thus reducing sink limitations on canopy photosynthesis at elevated CO2. Taken together, the data show the importance of maintaining strong demand from active sinks when the above-ground canopy is being exposed to elevated levels of the primary substrate of photosynthesis, CO2.

The small domain of cytochrome f from the psychrophile Chlamydomonas raudensis UWO 241 modulates the apparent molecular mass and decreases the accumulation of cytochrome f in the mesophile Chlamydomonas reinhardtii

Cytochrome f from the psychrophile Chlamydomonas raudensis UWO 241 has a lower thermostability of its c-type heme and an apparent molecular mass that is 7 kDa lower than that of the model mesophilic green alga Chlamydomonas reinhardtii. We combined chloroplast transformation, site-directed mutagensis, and the creation of chimeric fusion constructs to assess the contribution of specific domains and (or) amino acids residues to the structure, stability, and accumulation of cytochrome f, as well as its function in photosynthetic intersystem electron transport. We demonstrate that differences in the amino acid sequence of the small domain and specific charged amino acids in the large domain of cytochrome f alter the physical properties of this protein but do not affect either the thermostability of the c-type heme, the apparent half-life of cytochrome f in the presence of the chloroplastic protein synthesis inhibitor chloramphenicol, or the capacity for photosynthetic intersystem electron transport, measured as e-/P700. However, pulse-labeling with [14C]acetate, combined with immunoblotting, indicated that the negative autoregulation of cytochrome f accumulation observed in mesophilic C. reinhardtii transformed with chimeric constructs from the psychrophile was likely the result of the defective association of the chimeric forms of cytochrome f with the other subunits of the cytochrome b6/f complex native to the C. reinhardtii wild type. These results are discussed in terms of the unique fatty acid composition of the thylakoid membranes of C. raudensis UWO 241 adapted to cold environments.